1,3-Propanediol or trimethylene glycol is a valuable, but expensive chemical intermediate that is used as an additive to other substances or articles to enhance their physical properties or performance. 1,3-Propanediol is also used as a comonomer in the preparation of fiber and film-forming polymers. This chemical has found limited broad usage due to the high manufacturing costs associated with the feedstocks costs or the difficult process conditions.
Chemical preparation of 1,3-propanediol is known. For example ethylene oxide may be converted to 1,3-propanediol over a catalyst in the presence of phosphine, water, carbon monoxide, hydrogen and an acid. Alternatively 1,3-propanediol may be produced by the catalytic solution phase hydration of acrolein, or from hydrocarbons such as glycerol, reacted in the presence of carbon monoxide and hydrogen over catalysts from group VIII of the Periodic Table. These processes are energy intensive to run employing either high temperature or high pressure or both, resulting in a prohibitive costly process.
A microbiological or biochemical route to 1,3-propanediol, employing either metabolically-active microorganisms or the enzymes derived from biological sources, has been described. The process uses Enterobacter or Clostridium organism in a strict anaerobic habitat where glycerol is converted to 1,3-propanediol. The source or glycerol may be fossil fuels or from the water or residual waste stream from a distillery. Other organisms known to convert glycerol to propanediol are found e.g., in the species Klebsiella, Citrobacter, Clostridium, and Lactobacillus.
In addition to these native 1,3-propanediol producers recombinant organisms have also been constructed that convert glycerol to 1,3-propanediol. The genes responsible for the conversion of glycerol to 1,3-propanediol have been isolated and are all encompassed by the dha regulon. In order to make use of the advantages of higher protein expression and growth rate of recombinant bacteria, several attempts have been made to express the dha regulon as heterologous genes in E. coli. For example, the dha regulon from Citrobacter and Klebsiella have been expressed in E. coli and have been shown to convert glycerol to 1,3-propanediol. In one such system Tong et al., (Appl. Biochem. Biotech., 34, 149, (1992)) examined the improved production of 1,3-propanediol by cofermenting carbohydrates with glycerol.
In this system, a single organism uses the carbohydrate solely for a source of energy and enhanced cell growth. No propanediol was produced in the absence of exogenous glycerol. This study does not teach the conversion of carbohydrates into the carbon stream that produces 1,3-propanediol nor does it describe a mechanism for achieving this in a mixed culture as described in this Application.
Neither the chemical or biological methods described above for the production of 1,3-propanediol is well suited for industrial scale production since the chemical processes are energy intensive and the biological processes require the expensive starting material, glycerol. A method requiring low energy input and an inexpensive starting material is needed.
As with 1,3-propanediol, glycerol may be produced both by chemical and biological routes. Chemical processes generally employ petroleum-derived raw materials such as acrolein; allyl chloride; or propylene oxide and generally suffer from the same disadvantages as the chemical routes to 1,3-propanediol, including expensive raw materials or hazardous operating conditions.
Biological processes for the preparation of glycerol are known. The overwhelming majority of glycerol producers are yeasts but some bacteria, fungi and algae are also known. Bacteria, yeasts, and fungi produce glycerol by converting glucose or other carbohydrates through the fructose-1,6-bisphosphate pathway in glycolysis or the Embden Meyerhof Parnas pathway. Certain algae convert dissolved carbon dioxide or bicarbonate in the chloroplasts into the 3-carbon intermediates of the Calvin cycle. In a series of steps, the 3-carbon intermediate, phosphoglyceric acid, is converted to glyceraldehyde 3-phosphate which can be readily interconverted to its keto isomer, dihydroxyacetone phosphate, which is ultimately converted to glycerol. Although biological methods of both glycerol and 1,3-propanediol production are known, it has never been demonstrated that the two processes may be carried on together under the same reaction conditions. Such a process, utilizing mixed or linked cultures would represent an improvement in the production of 1,3-propanediol since it would be cost effective and would avoid the use of hazardous reagents.
The concept of successive or linked fermentations for biochemical transformations is known in the art and have been adapted for alcohol production. For example, Nakas et.al. (Appl. Environ. Microbiol. 46:1017-1023, 1983) describe a system for the production of mixed solvents of butanol, ethanol and 1,3-propanediol using a sequential fermentation process. The production system employs a photosynthetic algal genus, Dunaliella, to convert carbon dioxide to glycerol in a high salt medium. A Clostridium pasterianum strain was added to the CO.sub.2 -derived glycerol and algae mixture to produce a solvent blend that was primarily butanol.
The use of mixed cultures in industrial applications are known in the art but suffer from the requirement that each cell type be supported by a separate carbon substrate. So for example, yeast and lactic acid bacteria are used symbiotically in bread dough starter cultures. In these mixed systems there is no competition for the carbon substrate since the yeast uses only the glucose and the lactic acid bacterium uses only the maltose in the dough. Alternatively mixed culture systems have been developed where one organism produces a desirable effect in response to the presence of the other organism. So for example it has been demonstrated that in combinations of a bacterium (B. subtilis) with one of several yeasts, the induction of a bacterial protein was entirely dependent on the presence and concentration of a specific yeast in the medium. The increased sensitivity of yeasts to a specific class of antifungal agents when they are grown in mixed cultures with bacteria has been described.
Although applications of mixed cultures are known, it is a tenant of the art that the outcome in a mixed culture is not predictable. Mixed culture systems are particularly susceptible to complications caused by competition between organisms for the carbon source, diversion of the carbon out of the desired pathway, catabolite repression by the substrate, inhibition by the metabolites in the fermentation, and the difficulty in justifying the often highly dissimilar culture needs of each organism.
In spite of these difficulties in the use of mixed cultures, Applicants have succeeded in developing a mixed culture system that is capable of producing 1,3-propanediol from an unrefined carbohydrate source. Applicants have also devised a binary linked culture system for the production of 1,3-propanediol from a suitable carbon source. Applicants have overcome the difficulties of catabolite repression, feedback inhibition and carbon source diversion to create a system that is optimized for 1,3-propanediol production.